臺灣博碩士論文加值系統

中文摘要
本研究之目的，在於研究以蔗糖作為冷凍乾燥保護劑時，冷凍或冷凍乾燥過程對微脂粒及微脂粒/DNA複合物之轉染率的影響，以評估其發展成為長期安定製劑之可行性。此外，亦嘗試評估兩種膽固醇衍生物作為正電荷微脂粒脂質組成的可行性。
體外細胞株轉染實驗，使用老鼠之肝細胞株BNL CL.2作為轉染標的。以DC-chol(3β[N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol)和DOPE(dioleoyl phosphatidyl ethanolamine) 1:1（莫耳比）的比例，作為正電荷微脂粒的組成，並以本實驗室合成之 galactosyllipid修飾此正電荷微脂粒，製備Gal-liposome，以其作為基因遞送載體。新鮮之細胞株轉染配方的製備，乃是直接將定量的質體DNA（pGreen Lantern-1）以及定量的Gal-liposome混合。經冷凍或冷凍乾燥處理之轉染配方，乃是在新鮮之細胞株轉染配方中加入不同含量的蔗糖，再加以冷凍或冷凍乾燥。亦嘗試將加入不同含量蔗糖的Gal-liposome溶液，先行冷凍乾燥，而後重新水合並與質體DNA混合形成轉染配方。在安定性之評估時，乃將快速冷凍乾燥之Gal-liposome及Gal-liposome/DNA複合物（每0.268μmole total lipid含有1mmole蔗糖）儲存於-20℃及4℃，於冷凍乾燥後第三天、第七天及第二十八天重新水合製備成為轉染配方。將上述之各種轉染配方與細胞株BNL CL.2於細胞培養箱中培養5小時，再更換培養基成新鮮DMEM，繼續培養48小時，即可觀察結果。轉染效果之定量乃是使用流式細胞儀，計算表現出綠色螢光之細胞所佔比率。此外，以Pip-chol (4-methyl-piperazine-1-carboxy cholesterol)或Gua-chol ((2-guanidino-ethyl)-carbamoyl cholesterol)與DOPE以1:1（莫耳比）的比例混合，製成Pip-liposome及Gua-liposome，測量其zeta potential，以評估此二種膽固醇衍生物作為正電荷微脂粒脂質組成之效果。
本實驗之結果顯示，以每0.268μmole total lipid加入1mmole蔗糖作為冷凍保護劑之比例，可使得冷凍及冷凍乾燥Gal-liposome/DNA複合物保持與新鮮之細胞株轉染配方相當的轉染率。而在-20℃及4℃下儲存二十八天後之快速冷凍乾燥Gal-liposome及Gal-liposome/DNA複合物，仍具有與新鮮之細胞株轉染配方相當的轉染率。測量Pip-liposome及Gua-liposome之zeta potential發現，其表面並不具有正靜電荷。
總之，以冷凍乾燥處理並配合蔗糖作為冷凍乾燥保護劑，可以提供Gal-liposome及Gal-liposome/DNA複合物一種安定之製劑型態，但其更長期之安定性則需更進一步研究。而Pip-chol與Gua-chol則不適合與DOPE 作為正電荷微脂粒之組成。

Abstract
The main purpose of the present study was to evaluate the influence on the transfection efficiency of Gal-lipoosme/DNA complexes after freeze-thawing or freeze-drying in the presense of sucrose as cryoprotectant. Meanwhile, the possibility of two cholesterol derivatives to prepare cationic liposomes was also evaluated.
The mouse embryonic normal liver cell line (BNL CL.2) was used to be the transfection target. DC-chol (3β[N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol) and DOPE (dioleoyl phosphatidyl ethanolamine) (1/1 molar ratio) were selected for preparing the cationc DC-liposomes. DC-liposomes were modified by galactosyllipid which was synthesized in our laboratory (Gal-liposomes) and applied as a gene delivery vector. BNL CL.2 cells were incubated with fresh, freeze-thawed, or freeze-dried Gal-liposome/DNA complexes consisting of a reporter gene (pGreen Lantern-1) for 5 hrs in an incubator. Then the medium was replaced by fresh DMEM. After 48 hrs, the transfection efficiency was quantified by flow cytometery. To evaluate the stability of rapid freeze-dried Gal-liposomes and Gal-liposome/DNA complexes which were stored at —20℃ and 4℃, the transfection efficiency on the 3rd and 7th day after freeze-drying were also quantfied by flow cytometery. Pip-liposomes or Gua-liposomes were prepared by Pip-chol (4-methyl-piperazine-1-carboxy cholesterol)/DOPE or Gua-chol ((2-guanidino-ethyl)-carbamoyl cholesterol)/DOPE (1/1 molar ratio). Their ability to present on liposomes was evaluated by zeta potential values.
The results showed that freeze-thawed and freeze-dried Gal-liposome/DNA complexes were lyoprotected by 1mmole per o.268 μmole total lipid. The transfection efficiencies of rapid freeze-dried Gal-liposomes and Gal-liposome/DNA complexes stored at —20℃ and 4℃ after 28 days were not significant different from that of fresh Gal-liposome/DNA complexes. And according to zeta potential values, there was not net positive charge on the surface of Pip-liposomes or Gua-liposomes.
In summary, the rapid freeze-dried Gal-liposomes and Gal-liposome/DNA complexes could be stored under 4℃ for 28 days with entire transfection efficiency but the stability of extended storage period should be further studied. And Pip-chol and Gua-chol were not suitable to be the lipid content of cationic liposomes.

目錄
頁數
誌謝I
目錄II
附圖目錄V
附表目錄VIII
中文摘要IX
英文摘要XI
壹、緒論1
一、背景1
二、微脂粒簡介2
1.微脂粒之構造2
2.微脂粒的分類2
3.微脂粒與細胞的作用方式3
4.正電荷脂質6
三、肝臟細胞與去唾液酸胎醣蛋白接受器7
1.肝臟細胞7
2.去唾液酸胎醣蛋白接受器8
四、冷凍乾燥之原理與應用8
五、質體DNA：pGreen Lantern-110
1.Green fluorescence protein (GFP)10
2.pGreen Lantern-1的特點11
3.pGreen Lantern-1之優點11
六、流式細胞儀12
貳、研究動機與目的15
參、實驗材料與方法17
第一部份、純化質體DNA之大量抽取17
一、藥品與試劑17
二、耗材18
三、儀器及設備18
四、質體DNA18
五、實驗步驟19
第二部份、細胞培養24
一、藥品與試劑24
二、耗材24
三、儀器及設備25
四、細胞株25
五、實驗步驟26
第三部份、微脂粒之製備30
一、藥品與試劑30
二、耗材30
三、儀器及設備31
四、實驗步驟31
第四部份、微脂粒基因轉染配方之製備36
一、藥品36
二、耗材36
三、儀器及設備36
四、實驗步驟37
第五部份、細胞轉染實驗41
一、藥品41
二、耗材41
三、儀器及設備41
四、實驗步驟42
第六部份、資料處理與數據分析45
A.資料處理45
B.數據分析45
肆、結果與討論46
一、質體 DNA的抽取及確認46
抽取質體DNA的實驗部份46
質體DNA的確認部份47
二、微脂粒的製備48
Pip-liposome及Gua-liposome之製備48
脂質濃度測定50
微脂粒粒徑的測定51
微脂粒zeta potential的測定53
三、冷凍及冷凍乾燥產物之製備54
四、各種轉染配方之製備55
粒徑的測定55
zeta potential的測定56
五、轉染實驗58
轉染實驗的條件58
快速與慢速冷凍Gal-liposome/DNA複合物之轉染效果58
以冷凍乾燥Gal-liposome製備複合物之轉染效果59
快速慢速冷凍乾燥Gal-liposome/DNA複合物之轉染效果60
冷凍乾燥Gal-liposome及Gal-liposome/DNA複合物之安定性61
伍、結論63
陸、參考文獻64
柒、附圖73
捌、附表 108

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